Enzymes perform many of the most vital functions in our cells and tissues and are the targets of numerous transformative medicines. Considering the importance of enzymes in health and disease, it is both provocative and humbling to realize that the human proteome contains a huge number of uncharacterized enzymes. Assigning functions to these enzymes represents a grand challenge for researchers in the post-genomic era. To achieve this goal, selective pharmacological tools to perturb enzymes in living systems are needed. A pressing question, however, arises: how can one rapidly and systematically discover inhibitors for poorly characterized enzymes? Over the past decade, our lab has pioneered the development and application of an innovative chemical proteomic solution to this problem termed activity-based protein profiling (ABPP). The objective of this application is to use ABPP to discover potent, selective, and in vivo-active inhibitors for serine hydrolases (SHs), which are a large and diverse enzyme class that represents ~1% of all human proteins. SHs play critical roles in human physiology and disease and are targeted by several approved drugs. Despite their biological and biomedical importance, most SHs, including many with genetic links to human disease, lack inhibitors and consequently remain poorly understood with regards to their physiologic substrates and functions. In this this competitive renewal project, we will focus on inhibiting and functionally characterizing SHs with established or emergent roles in neurobiological processes. During the previous grant period, we created an efficient ABPP platform for SH inhibitor discovery and optimization that has already yielded selective and in vivo-active inhibitors for several SHs, as well as lead inhibitors for many additional enzymes. In most cases, these compounds represent the first pharmacological probes for studying their SH targets in living systems and are in widespread use by the biology research community. In this application, we propose to use a multidisciplinary research program that integrates ABPP with chemical synthesis, lipidomics, mouse genetics, and cell and animal pharmacology to selectively inhibit and functionally characterize: 1) SHs that regulate the biosynthesis of the endocannabinoid class of lipid transmitters in the nervous system (Specific Aim 1), and 2) SHs and SH pathways with genetic links to human neurological disorders (Specific Aim 2). We have enlisted a strong set of biology collaborators who will apply our optimized inhibitors to mouse models of nervous system function and disease. The inhibitors generated and knowledge gained herein should greatly advance our understanding of the functions of SHs and SH pathways, enabling the identification of drug targets to treat human neurological diseases.